improving allergen concentration homogeneity for investigating the effect of corona discharge on der...

Journal of Electrostatics 56 (2002) 43–53

Improving allergen concentration homogeneityfor investigating the effect of corona discharge on

Der p1 allergen

Neil Goodman*, John F. Hughes

Bioelectrostatics Research Centre, Department of Electronics and Computer Science, Faraday Building

(Building 21), University of Southampton, Highfield, Southampton, UK SO 17 1BJ

Received 8 June 2001; received in revised form 30 October 2001; accepted 1 November 2001

Abstract

Air ionisers utilising corona discharge have been commercially available for some time,

although clinical trials have shown that these do not improve the health of asthma sufferers.

New research has shown that the corona products can actually destroy allergens including the

main allergen of the house dust mite Dermatophagoides pteronyssinus; Der p1. Concentration

heterogeneity of commonly used sources of allergen and the additional problems that occur

with electrostatic studies mean that present methods of allergen preparation are unsatisfac-

tory. This paper describes work that improves the repeatability of tests investigating the effects

of ionic bombardment on Der p1 allergen concentration. Initial experimental methods

involved the distribution of sieved culture medium over a surface and divided into sectors for

concentration analysis but this gave extremely variable data and so any effect of an allergen

reduction method could not reliably be assessed. Different methods of distributing the sample

are described in this paper. The results show that by dissolving Der p1 samples and using small

evaporated aliquots, samples of homogenous concentration can be reliably used in electrostatic

investigations. The benefits of using the new evaporated solution method is described with a

comparison made between the effect of negative corona discharge on Der p1 using heterogeneous

Der p1-containing culture particles and the new technique described in the present paper. Using

the new technique of sample preparation, reductions in the Der p1 concentration increased from

a significant (Po0:01) 37.26% after 60min to 67.37% after 300min. Using faecal particles,

however, led to insignificant (P > 0:05) reductions that ranged from 4.11% to 26.86% after 60

and 300min, respectively. r 2002 Elsevier Science B.V. All rights reserved.

Keywords: Dust mites; Asthma; Air ionizer; Corona; Denaturing; Allergen

*Corresponding author. Tel.: +44-2380593703; fax: +44-2380593709.

E-mail address: [email protected] (N. Goodman).

0304-3886/02/$ - see front matter r 2002 Elsevier Science B.V. All rights reserved.

PII: S 0 3 0 4 - 3 8 8 6 ( 0 1 ) 0 0 2 0 3 - 0

1. Introduction

Over the past 20–30 years the incidence of bronchial asthma has steadily increasedin almost every country around the globe [1]. In parallel to this there have beenreports of up to 5-fold increases in the prevalence of house dust mites (HDMs) inrandom samples collected over a 10-year period from homes in both temperate [2]and warmer [3] climates. As the link between HDMs and the occurrence of asthmaand other atopic diseases is now well established [4], methods of reducing exposureto HDM allergens is of the utmost importance in the avoidance and control of thesediseases.Der p1 is the main allergen of the HDM Dermatophagoides pterynissinus. It is a

digestive enzyme that is secreted into the mite’s alimentary canal [5], which is thereason why 95% of all dust mite allergen is associated with the faecal particles [6].Der p1 is a protein that causes the cleavage of other proteins, the activity of which isthought to be related to its immunogenicity. In other words, Der p1 is an allergenpartly because of its functionality. As the protein is functional due to its tertiarystructure (which is due in part to electrostatic forces) the breakdown of this structureby altering, or interfering with its charges may modify its functionality. Allergen-carrying faecal particles have been found to tribo-charge predominantly to a positivepolarity [7]. This positive charge was retained even after prolonged contact with agrounded surface, which suggested that the charge was inherent to the proteincarried on the faeces. Analysis of the cDNA sequence has produced the sequence ofamino acids that make up the Der p1 protein [8]. If the number of positive andnegative charges is counted, it can be seen that the protein has a net +3 charge.Previous research into the use of air ionisers for asthma sufferers has shown little

to no evidence for any clinical benefit [9]. In some cases the condition of the patientwas even worsened [10]. It had been thought that the electrostatic precipitation ofairborne particulates by ionisers would benefit sufferers by reducing the level ofallergen and dust in the air leading to less being inhaled. However, the coronaproducts are harmful to airways and can lead to inflammation, worsening ofsymptoms and even prime the allergen-induced responses in the airways [11]. Newresearch has shown that corona products can be used to destroy allergens [12]. Witha revised protocol for use, air ionisers may have an application in the domesticenvironment to clean the house of allergy-causing proteins in situ before they canharm the body.To investigate the effects of corona discharge, a novel and improved method of

allergen sample preparation was required. The standard method of investigating theeffects of chemical or physical treatments on domestic allergens is to use house dust,HDM culture, or a mixture of both. This method has the advantage of mimicking insitu characteristics such as interactions with other dust components, and retention incarpeting and soft furnishings, etc. However, when investigating the effects of eithera chemical or physical treatment on allergens, the use of heterogeneous powdermixtures inevitably leads to concentration variability between samples. Suchvariability means that the exact concentration of the samples tested cannot beknown precisely. This could lead to difficulties in distinguishing the effects of any

N. Goodman, J.F. Hughes / Journal of Electrostatics 56 (2002) 43–5344

compound or technique on the concentration of the allergen in the sample, and evenlead to false results. Spurious effects can be recorded using this method, such as anincrease in concentration of the allergen in a sample that has been treated with atechnique known to reduce the allergen (see for example, [13]). Consequently, a largenumber of replicates are typically necessary in order to discern any statisticallysignificant difference between samples and controls. This leads to an expense in bothtime and resources.Powdered samples are also sensitive to accidental loss to the surrounding

environment during handling. Added difficulties arise if the effects of electrostatictechniques, in particular corona discharge, on the allergen are being investigated asdescribed below. When testing novel techniques of destroying harmful allergens invitro a more reliable method of preparing samples of known concentration is needed.This paper investigates the best method of preparing allergen samples for the

analysis of any effects that corona discharge may have upon the allergen. Thevariability of Der p1 concentration in samples of sieved, concentrated Dermatopha-

goides pteronyssinus HDM culture using different methods of application wasmeasured. The effect of negative corona discharge on Der p1 using theheterogeneous Der p1-carrying faecal particles and the new evaporated solutionmethod are also described and compared.

2. Materials and methods

2.1. Preparation of HDM cultures

Cultures containing Dermatophagoides pterynissinus HDMs were established byusing material obtained from British domestic vacuum cleaner bags that testedpositive for Der p1. Cultures were maintained on a substrate of finely crushed dogbiscuit and dried yeast mixed in a ratio of approximately 30 to 1 [14]. The establishedHDM culture was then sieved to below 63 mm.

2.2. Distributing culture medium particles with a spatula or sieve

A circular piece of aluminium foil (70mm diameter) was divided equally into eightsectors. One gram of sieved culture medium was then spread as evenly as possibleover the foil using a spatula. The culture medium in the individual sectors was thenbrushed into an eppendorf tube and 1% BSA-PBS-T (Bovine Serum Albumin in0.05% Phosphate Buffered Saline with Tween20) was added to produce a 1 in 100dilution for subsequent testing for Der p1 concentration by a two-site monoclonalantibody enzyme-linked immunosorbent assay [15] (ELISA, from INDOORBiotechnologies Ltd., Cardiff, UK). This procedure was repeated twice. Resultsare shown in Fig. 2A.To test the importance of the dust distribution technique, 1 g of sieved culture

medium was evenly distributed over the foil circles using a fine sieve (63 mm pore

N. Goodman, J.F. Hughes / Journal of Electrostatics 56 (2002) 43–53 45

size), and then removed as above and prepared for ELISA. This was also repeatedtwice. Results are shown in Fig. 2B.

2.3. Using an aqueous solution of culture medium

Another application method employed an aqueous solution of the culturemedium. Fifteen grams of the o63 mm culture fraction was added to 400ml ofdistilled water. This mixture was stirred well to enable all the Der p1 to dissolve, thenpassed through filter paper (Whatman, qualitative, grade 4) to remove solid material.Thimerosal (sodium ethylmercurithiosalicylate) was added at a concentration of0.001% to prevent bacterial, fungal and mould growth. Three hundred millilitres ofthis solution were then poured onto an aluminium foil-covered 736 cm2 tray anddried at 371C. Two circular pieces of foil of 70mm diameter were then cut out of thefoil at random positions, each were divided into eight sectors and placed in aneppendorf. One per cent BSA-PBS-T was added to give a 1 in 200 dilution inpreparation for testing by ELISA. Results are shown in Fig. 2C.The final method of Der p1 application investigated also used the culture medium

solution, prepared as above. Sixteen 100 ml aliquots of this solution weremicropipetted onto foil and left to evaporate in a drying oven at 371C. These werediluted 6-fold with 1% BSA-PBS-T and prepared for ELISA. Results shown inFig. 2D.In order to determine whether the act of evaporating the aqueous solution of Der

p1 had any detrimental effect on the concentration itself, 12 100 ml samples wereevaporated at 371C and diluted in 1% BSA-PBS-T as above and then assayed byELISA. These samples were then compared to the Der p1 concentration of 12 100 mldrops that were placed straight into an eppendorf and diluted 6-fold in 1% BSA-PBS-T without being evaporated.

2.4. Exposing Der p1-carrying faecal particles to negative continuous glow corona

discharge

A simple point-to-plane corona apparatus was used with a pin with point radius45 mm fixed with an interelectrode gap of 15mm above a square, planar aluminiumelectrode. This is illustrated schematically in Fig. 1. Onto this, 400 mg of finely sieved(o63 mm) culture medium was placed using a spatula and half of this was thenremoved, weighed and kept as the control in ambient air for the duration of theexperiment. The remaining sample was then distributed evenly over the planarelectrode. The pin was connected to a negative d.c. high voltage supply to producean ionic bombardment equivalent to a corona current of 90 mA. Samples wereexposed for 60, 120, 180, 240 and 300min. The sample was then carefully removed,weighed and diluted 100-fold with 1% BSA-PBS-T in preparation for ELISA. Thisprotocol was followed to produce 12 replicates. Any solid particulates thataccumulated on the pin during corona discharge was brushed into an eppendorfand added to 200ml 1% BSA-PBS-T and prepared for ELISA. Results shown inFig. 3.


2.5. Exposing evaporated Der p1 solution to negative continuous glow corona

discharge

A solution containing HDM allergen was prepared as described in Section 2.3.Hundred-microlitre samples of this Der p1 solution were then pipetted onto 20mm2

squares of aluminium foil. The sample and its paired control were preparedadjacently. These were then dried at 371C. The Der p1 sample was then fixed to theplanar electrode and exposed to corona discharge as described in Section 2.4. Afterthis exposure time the sample was removed and prepared for ELISA. The controlswere exposed to ambient air for the equivalent period of time, diluted 6-fold in 1%BSA-PBS-T and then prepared in the same way. This was repeated 6 times for eachexposure. Results are shown in Fig. 3.

2.6. Data analysis

The concentration data from the solid mixture of culture medium distributed byspatula or sieve (Section 2.2) were expressed as mg g�1 of culture medium, and thedata from the tests using culture medium solution (Section 2.3) were expressed asngml�1. The variability of the samples was then compared using the coefficient ofvariation (CV), which is the ratio of the standard deviation to the mean, expressed asa percentage.For each duration of exposure to corona (Sections 2.4 and 2.5), the Der p1

concentrations of the exposed samples were compared to their paired controls by theF-test. The concentration data for each time period was then analysed using thePaired t-test for means, or the Mann Whitney-U test where appropriate. Statisticalsignificance was defined as Po0:05: These concentration data were then presented asmean percentage reductions in Der p1 concentration calculated by comparing theDer p1 sample to its equivalent control in the specific time period.

Fig. 1. Schematic diagram of the point-to-plane corona apparatus.


3. Results

3.1. Concentration homogeneity

The Der p1 concentration can be seen to vary considerably in all tests using sievedculture medium. Fig. 2 shows the Der p1 concentration of the individual samplestaken from the sectors of the foil and also of the aliquots of culture medium solution.Fig. 2A shows the concentration of sectors with allergen sample distributed with aspatula. The concentration of most sectors varied from 32.45 to 99.63 mg g�1 butsectors 7 and 8 had a much greater concentration of 604.81 and 618.81 mg g�1,respectively.

0

100

200

300

400

500

600

700

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Sector Number

[Der

p1]

/ µgg

-1

0

10

20

30

40

50

60

70

80

90

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Sector Number

(A)

(B)

[Der

p1]

/ µgg

-1

Fig. 2. The concentration of Der p1 in the individual samples when distributed using various methods of

application. (A) Using a spatula to distribute the sieved culture; (B) using ao63 mm pore sieve; (C) using aDer p1 solution evaporated onto a tray; (D) using 100ml aliquots, notice the uniformity of concentrationin these samples compared to the other methods above.


Using a sieve to spread the allergen samples (see Fig. 2B) led to Der p1concentrations that varied from 5.91 to 83.27 mg g�1. Allowing 300ml of the Der p1solution to evaporate on foil led to a greater variability in concentration than using asieve to distribute solid particles (see Fig. 2C). The Der p1 concentration varied from6.04 to 64.80 mg g�1.However, using the evaporated 100 ml drops of Der p1 solution led to a very

uniform concentration throughout the samples tested (see Fig. 2D). The concentra-tion of samples varied from 54.43 to 77.03 ngml�1; a range of only 22.60 ngml�1

which is an acceptable variability and corresponds to the uniform nature ofhomogeneous solutions. Table 1 gives the coefficient of variation between sectors foreach of the methods of sample application. These values show that the concentrationvariability of a Der p1 sample distributed by a spatula is the greatest followed by theevaporating 300mls of Der p1 solution onto a tray method, using a sieve and the

0

10

20

30

40

50

60

70

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Sector Number

0

10

20

30

40

50

60

70

80

1 9 10 11 12 13 14 15 16

Sample number

(C)

(D)

[Der

p1]

/ ngm

l-1

2 3 4 5 6 7 8

[Der

p1]

/ ngm

l-1

Fig. 2. (Continued).


aliquot method varied least. The 100 ml aliquot method gave a concentrationvariation of more than 10-fold less than using a spatula to distribute the sample. Theresults from the experiment to determine whether evaporation reduced theconcentration of Der p1 showed no difference between the two sets of samples.

3.2. Effect of corona discharge on Der p1

Fig. 3 shows that exposure of evaporated Der p1 samples to negative coronadischarge (see Section 2.5) led to statistically significant amounts of Der p1 beingdenatured with six replicates of each time point (Po0:01; using the Paired t-test formeans). The percentage reductions in Der p1 concentration increased from37.2676.47% after 60min of exposure through 39.8077.49%, 43.2277.07%,55.8177.58% and 67.3775.78% reduction after 120, 180, 240 and 300min exposureto negative continuous glow corona, respectively.Similar to the results obtained from using evaporated Der p1 samples, the mean

percentage reductions in the Der p1 concentration of dry culture medium (seeSection 2.4) increased with each increase in exposure time (see Fig. 3). Thepercentage reductions increased from 0.8711.05% after 60min exposure through4.1177.07%, 19.8379.45%, 21.22712.46% and 26.86714.64% after 120, 180, 240and 300min exposure, respectively.However, as would be expected from the large standard errors of these results,

these reductions were not statistically significant. This reinforces the argument thatthe use of heterogeneous powder mixtures inevitably leads to concentrationvariability and difficulties in distinguishing the effects of any technique upon thesample.Another problem encountered during corona discharge on the dry culture medium

was the loss of sample to the surrounding environment. Electrostatic charging andfield effects led to a visible accumulation of solid material on the upper electrode.The total amount was small, weighing o1mg. However, by adding this to 200 mlof 1% BSA-PBS-T, the ELISA determined that the sample consisted of 23.92 ng ofDer p1.

4. Discussion

This investigation has demonstrated that the inherent heterogeneity of sieved,allergen containing samples causes significant variability in data obtained from

Table 1

The coefficient of variation of the samples’ concentration taken from the experiments using different

methods of Der p1 distribution

Method of sample application Spatula Sieve Tray Aliquot

CV 146.07 55.55 77.04 10.82


them. Additional problems encountered when using a powdered sample in theelectrostatic investigations demonstrated here were the loss of material to theatmosphere and electrodes. The particles of the powder mixture on the planarelectrode became electrostatically charged as a consequence of exposure to uni-polarions. Particle movement within the interelectrode gap was subsequently induced bythe electric field. This explains the behaviour of the powder escaping the corona set-up or being attracted to the negative polarity of the upper electrode. The allergencould also have been attracted to the opposite polarity of the upper electrode becauseof its inherent net positive polarity in dry conditions [7]. These artefacts caused the12 replicates to be insufficient in obtaining a statistically significant value for theamount of Der p1 denatured.The use of single, small aliquots taken from a dissolved sample has been shown to

overcome these problems and is ideal for investigating the effect of electrostatictechniques on protein samples. Consequently, the consistency and reliability ofallergen denaturing tests can be substantially improved. The sample is firmlyattached to the planar electrode and so will not be lost to the atmosphere, normigrate to the upper electrode. This method also allows different electrodeconfigurations to be tested such as wire-to-cylinder and wire-to-plane as the samplesmay also be placed vertically or inverted. This method of allergen preparation wouldalso be suitable for future experimental procedures for evaluating the effects ofelectric fields on allergens.

Exposure Time/ min

60 120 180 240 300

Mea

n %

Red

uctio

n in

[Der

p1]

-10

0

10

20

30

40

50

60

70

Fig. 3. The mean percentage reduction in Der p1 concentration after irradiation by negative continuous

glow corona on sieved culture medium (�), or aliquots of evaporated Der p1 solution (J). Culturemedium: n ¼ 12; evaporated solution: n ¼ 6; SEM shown.


With just six replicates for each time of exposure of evaporated samples tonegative glow corona, all percentage reductions in Der p1 concentration weresignificant and with small variation about the mean. The percentage reductionsobserved in the concentration of Der p1 in the evaporated samples were considerablyhigher than those observed with the sieved culture. This could be due to a number offactors, such as loss of non-Der p1 material through handling the powder material orthrough electrostatic attraction to the pins. When using a pin-to-plane coronaarrangement, the point electrode became thinly coated with the allergenic sample. Thiswould further alter the allergen concentration of the sample on the planar electrodegiving a false reduction in the final analysis. Another possibility is that the non-allergenicparticles of the solid mixture physically protect the Der p1 molecule from exposure to thecorona products. Research is ongoing into the possibility of a relationship between theconcentration of the sample and the percentage reduction observed.The tests reported here concentrated on the Der p1 allergen specifically. Other

dust mite allergens including Der p2, Der f1 and the major cat allergen Fel d1 arecurrently under investigation using the same allergen preparation technique [16].With the discovery that corona discharge can destroy Der p1 and other allergens,intense research efforts will be focussed into producing an optimised technique andprotocol for applying corona discharge to destroy allergens in the domesticenvironment. The improved method for preparing allergen samples detailed here willaid these efforts.

Acknowledgements

The support of the National Asthma Campaign, who funded this work, is gratefullyacknowledged.

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